Recent Clinical Advances on Long Non-Coding RNAs in Triple-Negative Breast Cancer

Triple-negative breast cancer (TNBC) is a more aggressive type of breast cancer due to its heterogeneity and complex molecular mechanisms. TNBC has a high risk for metastasis, and it is difficult to manage clinical conditions of the patients. Various investigations are being conducted to overcome these challenges using RNA, DNA, and proteins for early diagnosis and treatment. Recently, long non-coding RNAs (lncRNAs) have emerged as a novel target to treat the multistep process of TNBC. LncRNAs regulate epigenetic expression levels, cell proliferation and apoptosis, and tumour invasiveness and metastasis. Thus, lncRNA-based early diagnosis and treatment options could be helpful, especially for patients with severe TNBC. lncRNAs are expressed in a highly specific manner in cells and tissues and are involved in TNBC progression and development. lncRNAs could be used as sensitive and specific targets for diagnosis, treatment, and monitoring of patients with TNBC. Therefore, the exploration of novel diagnostic and prognostic biomarkers is of extreme importance. Here, we discuss the molecular advances on lncRNA regulation of TNBC and lncRNA-based early diagnosis, treatment, and drug resistance.


Introduction
Breast cancer (BC) is caused by epigenetic modifications and is a highly heterogeneous disease. It exhibits various histological and clinical features [1]. There are five main intrinsic molecular subtypes of BC. To my knowledge, the five intrinsic molecular subtypes are: Luminal A, Luminal B, HER2-enriched, Triple-negative/Basal-like, and Claudin-low. Among these, Luminal A breast cancers are HER2-negative (HR+/HER2−) and include ER+/PR+, ER+/PR−, and ER−/PR+ status. (Figure 1) [2]. Triple-negative breast cancer (TNBC) is the most aggressive molecular clinical subtype of all invasive cancers. Various genetic markers are expressed during the development of TNBC ( Figure 2) [3], which are involved in a gain or loss of function. Genetic markers involved in gain of function include EGFR, EGFR1/2, VEGFR, MYC, AR, CCNE, MDM2, PIK3CA, MAGI-AKT3, MYO3A, PARK2, and genetic markers involved in loss of function are INPP4B, PTEN, CDKN2A, BRCA1/2, TP53, RB1, and MLL3 ( Figure 2) [4]. Advances in genetic heterogeneity research have revealed prognostic and therapeutic targets in TNBC [1][2][3]. TNBC has been divided into four distinct subtypes using gene expression analysis: basal-like immune-activated (BLIA), basal-like immune-suppressed (BLIS), mesenchymal (MES), and luminal androgen receptor (LAR). The classification of TNBC subtypes lacks a standardised system, however. Basal-like (BL) tumours make up around 80% of TNBC tumours, and because BL tumours cluster physiologically apart from the other BC subtypes, intrinsic subtyping is less effective for significant diagnosis and classification than it is for the other clinical subtypes. Multiple initiatives to investigate DNA, RNA, microRNA, and protein expression patterns through cross-platform

LncRNAs
lncRNAs are actively involved in gene expression, epigenetic deregulation, chromatin remodelling, DNA methylation, translation of oncogenic gene targets, and biogenesis ( Figure 3). They are transcribed by RNA polymerase II, after which most transcripts are spliced, and are mainly found in the nucleus and chromatin, being expressed in cells and

LncRNAs
lncRNAs are actively involved in gene expression, epigenetic deregulation, chromatin remodelling, DNA methylation, translation of oncogenic gene targets, and biogenesis ( Figure 3). They are transcribed by RNA polymerase II, after which most transcripts are spliced, and are mainly found in the nucleus and chromatin, being expressed in cells and

LncRNAs
lncRNAs are actively involved in gene expression, epigenetic deregulation, chromatin remodelling, DNA methylation, translation of oncogenic gene targets, and biogenesis ( Figure 3). They are transcribed by RNA polymerase II, after which most transcripts are spliced, and are mainly found in the nucleus and chromatin, being expressed in cells and tissues in a specific manner [6,9,17]. Transcriptional regulation and various molecular processes in the cytoplasm are controlled by lncRNAs; various circulating lncRNAs are transmitted via exosomes and bind to various transcription factors, chromatin-regulated complexes, RNA-binding proteins, nascent RNA transcripts, and chromatin [17]. The normal expression of lncRNAs and the effect of their expression changes on tumour behaviour depends on the canonical function of the mRNA target genes (Figure 4). lncRNAs can bind to the active site of proteins and regulate molecular processes at the post-transcriptional level. They are involved in functional biological processes at the cellular or physiological levels. RNA-induced silencing complexes (RISCs) are formed with the help of lysine-specific demethylase 5B (KDM5B, also known as histone demethylase JARID1B), trimethylation of lysine 4 on the histone H3 protein subunit (H3K4me3), monomethylation of lysine 4 on the histone H3 protein subunit (H3K4me1), hsa-miR-448 (also known as miRNA448), breast cancer 1/2 (BRCA1/2), retinoblastoma protein (pRB), caveolin-1 (CAV-1), Homeobox protein Hox-A5 (HOXA5), Stratifin (SFN), methyl groups (CH3), and Ras homolog gene family, member A (RhoA) (Figures 3 and 5) [18]. In 2019, it was found that the lncRNA MIR100HG regulates proliferation in TNBC and the expression of the p27 gene after formation of an RNA-DNA triplex at the promoter [19]. Moreover, MIR100HG silencing leads to reduced transcription and translation of p27 [19,20]. Three triplex-forming oligonucleotides (TFOs) have been observed on the lncRNA of p27, which binds to the triplex-targeting ability (TTA) site at the 5'UTR; this event has been observed in TNBC cell lysates [21]. The binding of TFO1 and TTA is a unique mechanism by which MIR100HG regulates the transcription factors at the promoter region of p27 [21,22]. Plasmacytoma variant translocation 1 (PVT1) is another type of lncRNA that is transcribed by a gene situated at the 8q24 chromosomal region and plays and important role in TNBC development. It contains 12 exons that when spliced generate lncRNAs [23]. PVT1 binds to Krüppel-like factor 5 (KLF5) and generates a BAP1 deubiquitinase that induces TNBC via beta-catenin upregulation. Furthermore, the PVT1 promoter also acts as a regulator of the expression of the MYC proto-oncogene and BHLH transcription factor (c-MYC) [24]. These findings show that lncRNAs also mediate regulation at the transcriptional level. tissues in a specific manner [6,9,17]. Transcriptional regulation and various molecular processes in the cytoplasm are controlled by lncRNAs; various circulating lncRNAs are transmitted via exosomes and bind to various transcription factors, chromatin-regulated complexes, RNA-binding proteins, nascent RNA transcripts, and chromatin [17]. The normal expression of lncRNAs and the effect of their expression changes on tumour behaviour depends on the canonical function of the mRNA target genes ( Figure 4). lncRNAs can bind to the active site of proteins and regulate molecular processes at the post-transcriptional level. They are involved in functional biological processes at the cellular or physiological levels. RNA-induced silencing complexes (RISCs) are formed with the help of lysine-specific demethylase 5B (KDM5B, also known as histone demethylase JARID1B), trimethylation of lysine 4 on the histone H3 protein subunit (H3K4me3), monomethylation of lysine 4 on the histone H3 protein subunit (H3K4me1), hsa-miR-448 (also known as miRNA448), breast cancer 1/2 (BRCA1/2), retinoblastoma protein (pRB), caveolin-1 (CAV-1), Homeobox protein Hox-A5 (HOXA5), Stratifin (SFN), methyl groups (CH3), and Ras homolog gene family, member A (RhoA) (Figures 3 and 5) [18]. In 2019, it was found that the lncRNA MIR100HG regulates proliferation in TNBC and the expression of the p27 gene after formation of an RNA-DNA triplex at the promoter [19]. Moreover, MIR100HG silencing leads to reduced transcription and translation of p27 [19,20]. Three triplex-forming oligonucleotides (TFOs) have been observed on the lncRNA of p27, which binds to the triplex-targeting ability (TTA) site at the 5'UTR; this event has been observed in TNBC cell lysates [21]. The binding of TFO1 and TTA is a unique mechanism by which MIR100HG regulates the transcription factors at the promoter region of p27 [21,22]. Plasmacytoma variant translocation 1 (PVT1) is another type of lncRNA that is transcribed by a gene situated at the 8q24 chromosomal region and plays and important role in TNBC development. It contains 12 exons that when spliced generate lncRNAs [23]. PVT1 binds to Krüppel-like factor 5 (KLF5) and generates a BAP1 deubiquitinase that induces TNBC via betacatenin upregulation. Furthermore, the PVT1 promoter also acts as a regulator of the expression of the MYC proto-oncogene and BHLH transcription factor (c-MYC) [24]. These findings show that lncRNAs also mediate regulation at the transcriptional level.

Clinical Updates on lncRNAs in TNBC
Recently, lncRNA expression in patients with TNBC was investigated; 1034 lncRNAs were identified using NGS technologies and microarrays, out of which, 537 lncRNAs regulate 451 protein-coding genes [14]. These genes are also detected in TNBC cells and are

Clinical Updates on lncRNAs in TNBC
Recently, lncRNA expression in patients with TNBC was investigated; 1034 lncRNAs were identified using NGS technologies and microarrays, out of which, 537 lncRNAs regulate 451 protein-coding genes [14]. These genes are also detected in TNBC cells and are

Clinical Updates on lncRNAs in TNBC
Recently, lncRNA expression in patients with TNBC was investigated; 1034 lncRNAs were identified using NGS technologies and microarrays, out of which, 537 lncRNAs regulate 451 protein-coding genes [14]. These genes are also detected in TNBC cells and are involved in cell signalling pathways such as the MAPK and PI3K-Akt pathways, which may lead to heterogeneity [14,24]. lncRNAs also act as miRNAs, binding to miRNA-targeted mR-NAs and dysregulated miRNAs [25]. This crosstalk forms a complex post-transcriptional regulatory network including mRNAs and lncRNAs that is called the competing endogenous RNA (ceRNA) network [26]. ceRNA-mediated regulatory mechanisms constitute an important pathway in lncRNA-modulated post-transcriptional regulation in TNBC [27]. A microarray-based ceRNA network analysis revealed that 4852 lncRNAs are related to the diagnosis and treatment outcome of TNBC [28]. Another study using the TCGA database found that 150 lncRNAs are expressed at the tissue level and 823 in serum and these lncRNAs could act as prognostic factors in TNBC [29]. Furthermore, the study found that the lncRNA OSTN-AS1 is a novel immune-related prognostic marker [29]. An integrated ceRNA network involving three miRNAs (CHRDL1, FCGR1A, and RSAD2) and two lncR-NAs (HIF1A-AS2 and AK124454) was developed using microarray analysis [30]. These findings demonstrate that lncRNAs play major roles in the regulation of cell signalling, genetic heterogeneity, TNBC development, and pathological features ( Figure 6) shown in Table 1. involved in cell signalling pathways such as the MAPK and PI3K-Akt pathways, which may lead to heterogeneity [14,24]. lncRNAs also act as miRNAs, binding to miRNA-targeted mRNAs and dysregulated miRNAs [25]. This crosstalk forms a complex post-transcriptional regulatory network including mRNAs and lncRNAs that is called the competing endogenous RNA (ceRNA) network [26]. ceRNA-mediated regulatory mechanisms constitute an important pathway in lncRNA-modulated post-transcriptional regulation in TNBC [27]. A microarray-based ceRNA network analysis revealed that 4852 lncRNAs are related to the diagnosis and treatment outcome of TNBC [28]. Another study using the TCGA database found that 150 lncRNAs are expressed at the tissue level and 823 in serum and these lncRNAs could act as prognostic factors in TNBC [29]. Furthermore, the study found that the lncRNA OSTN-AS1 is a novel immune-related prognostic marker [29]. An integrated ceRNA network involving three miRNAs (CHRDL1, FCGR1A, and RSAD2) and two lncRNAs (HIF1A-AS2 and AK124454) was developed using microarray analysis [30]. These findings demonstrate that lncRNAs play major roles in the regulation of cell signalling, genetic heterogeneity, TNBC development, and pathological features ( Figure  6) shown in Table 1.

Importance of lncRNAs in Treatment
lncRNAs affect the response to treatments such as chemotherapy, immunotherapy, and radiotherapy [99]. H19 is expressed in patients with TNBC during neoadjuvant chemotherapy and is related to effective clinical outcomes. LINK-A expression is linked to response to pembrolizumab treatment in patients with TNBC because its decreased expression reduces CD8 + T-cell infiltration [59]. These lncRNAs act as biomarkers for treatment response in patients with TNBC. LncAFAP1-AS1 expression has been observed in patients with TNBC who received radiotherapy after surgery, and this lncRNA acts as biomarker for radiotherapy [82]. Moreover, lncRNAs are involved in angiogenesis. LINC01133 expression is induced by mesenchymal stem/stromal cells that adjoin TNBC cells [33]. lncRNAs are actively involved in the regulation of cell proliferation and apoptosis as well as drug resistance in TNBC [16,44,47,61,99]. DRHC and HOTAIR inhibit TNBC growth and development [31]. HOTAIR plays a role in the invasion and migration of TNBC cells and is used as a biomarker for TNBC metastasis in circulation and tissues, indicating poor survival and response [31,32]. DRHC inhibits TNBC cell proliferation by downregulating the expression of HOTAIR, whereas HOTAIR does not affect the expression level of DRHC. H19 expression is reduced in TNBC cells, whereas PTCSC3 expression is not altered by H19 overexpression [61]. HIST2H2BC and SNRPEP4 were identified in 165 frozen tissue samples by transcriptome microarrays; these lncRNAs are involved in taxane chemotherapy in patients with TNBC. Increased miR-377-3p expression delays TNBC progression by regulating the inc00339/miR-377-3p/HOXC6 axis and inhibits TNBC proliferation and apoptosis. Therefore, it is used as therapeutic target. HIF1A-AS2 expression is upregulated in TNBC mammary tissue, which is linked to overall survival. HOTAIR is closely associated with androgen receptor expression and used as a therapeutic strategy to prevent metastasis. The miR-199a/FOXP2 pathway is induced by LINC01133 and triggers the proliferation of TNBC cells. Various lncRNAs act as stem cell markers, such as DANCR, LINC01638, LINC-ZNF469-3, NEAT1, NRAD1, and ASRPS [75,87]. Some lncRNAs promote vasculogenic mimicry, providing growth supplementation for tumour formation in TNBC. TP73-AS1, which is activated by the miR-490-3p/TWIST1 pathway, is one example. LINK-A alters glycolysis by mediating HIF1α phosphorylation at Tyr565 and Ser7 [3,16,44,47]. MANCR inhibits DNA damage and prevents disease progression [66]. AWPPH is involved in the prevention of tumourigenesis upon treatment with carboplatin; AWPPH small interfering RNA (siRNA) silencing leads to increased chemosensitivity in TNBC [10,56]. TUG1 induces the expression of miR-197, reduces the activation of WNT signalling, and enhances TNBC cell sensitivity to cisplatin [75]. These findings demonstrate the importance of lncRNAs in the prevention of tumourigenesis. More studies are required to explore lncRNA treatment options. Early studies showed that HOTAIR recruits the polycomb repressive complex 2 to its target genes through the CoREST/REST H3K4 demethylase complex [75].

Nanoparticle-Based Targeted Therapy with ncRNAs for TNBC
lncRNAs are versatile, able to exert multilevel gene regulation, and have emerged as therapeutic targets for clinically complicated TNBC cases ( Figure 6) [25]. DANCR may potentially be used to reduce the limitations of monotherapy in TNBC networks and to lower the risk of side effects in healthy tissues [58]. DANCR is targeted by RNA interference (RNAi) (Figures 5 and 6). The main challenge of RNAi therapy is targeted delivery; a non-viral siRNA-based delivery system has shown limited efficacy and temporary expression [94]. Amino acid-based lipid carriers have shown promising results in siRNA, nucleic acids, and CRISPR/Cas-based approaches [95]. DANCR overexpression was established in TNBC using RGD-PEG-ECO/siDANCR nanoparticles for effective cytosolic delivery of siDANCR [96]. Injection of the RGD-PEG-ECO/siDANCR nanoparticles led to reduce the progression of disease severity (Figure 7) [97]. In another study, LINC00511-siRNA was used to deliver siRNA in patients with TNBC [97]. Nanoengineered platforms were used to deliver lncAFAP1-AS1 siRNA (siAFAP1-AS1) to reverse radioresistance and increase the efficacy in TNBC tumour models [98]. These findings demonstrate that RNA nanoparticlebased targeted therapy can be more effective in TNBC. In conclusion, various lncRNAs are abnormally expressed and used in the treatment of TNBC, including ASOs, LNA, or RNA nanotechnology targeting lncRNAs.
reduce the progression of disease severity (Figure 7) [97]. In another study, LINC00511-siRNA was used to deliver siRNA in patients with TNBC [97]. Nanoengineered platforms were used to deliver lncAFAP1-AS1 siRNA (siAFAP1-AS1) to reverse radioresistance and increase the efficacy in TNBC tumour models [98]. These findings demonstrate that RNA nanoparticle-based targeted therapy can be more effective in TNBC. In conclusion, various lncRNAs are abnormally expressed and used in the treatment of TNBC, including ASOs, LNA, or RNA nanotechnology targeting lncRNAs.

LncRNAs Involved in The Regulation of Drug Resistance
lncRNAs can alter the genetic regulation that may lead to the development of drug resistance. GAS5 promotes drug resistance to adriamycin, paclitaxel, and cisplatin [3,16,44,47]. Furthermore, GAS5 expression is reduced by mTORC1/mTORC2 (AZD8055) and PI3K/mTOR (BEZ235) inhibitors [99]. HOTAIR expression is controlled by EGFR/HER-2 inhibitor-based treatment such as lapatinib or the c-ABL inhibitor imatinib. Suppression of HOTAIR expression by the action of β-catenin on the HOTAIR promoter at the LEF1/TCF4-binding site increases drug resistance to combined therapy [100]. LINC01139 binding to the pleckstrin homology domain of AKT leads to hyperactivation and causes drug resistance to AKT inhibitors, which are commonly used for treatment of patients with TNBC [101]. Another treatment option for TNBC is immunotherapy-based treatments. LINK-A causes drug resistance by activating LINK-A-PKA-TRIM71 signalling, reducing the efficacy of immune checkpoint inhibitor-based treatments [102]. HIF1A-AS2 and AK12 4454 also cause drug resistance in TNBC cells [30]. H19 and NEAT1 induce resistance to paclitaxel through the AKT signalling pathway [16,61]. BORG causes resistance to doxorubicin through NF-κB signalling ( Figure 8) [4,74]. DNA damage and repair is a complex process, and various signalling pathways are involved; impairment of this process leads to tumour development [103]. LINP1 participates in DNA double-strand break repair mechanisms using a scaffold linked up with Ku80 and DNA-PKcs, which prevents resistance to ionizing radiation [104]. INP1 prevents resistance to radiotherapy in TNBC [105]. The knockout of PCAT6 enhances the radiosensitivity of TNBC cells via the miR-185-5p/TPD52 axis [50]. These findings demonstrate the potential use of lncRNAs for the regulation of drug resistance in patients with TNBC (Table 2 and Figure 8). Further studies are required to investigate the roles of other lncRNAs in drug resistance.
which prevents resistance to ionizing radiation [104]. INP1 prevents resistance to radiotherapy in TNBC [105]. The knockout of PCAT6 enhances the radiosensitivity of TNBC cells via the miR-185-5p/TPD52 axis [50]. These findings demonstrate the potential use of lncRNAs for the regulation of drug resistance in patients with TNBC (Table 2 and Figure  8). Further studies are required to investigate the roles of other lncRNAs in drug resistance.

Future Perspectives of lncRNAs as Potential Diagnosis and Pharmacological Tools/Targets
Different aspects of lncRNAs remain unclear, including their expression patterns in TNBC cells and their role in the modulation of mRNA coding genes [14,25]. Understanding their molecular heterogeneity would be helpful for developing novel drugs [113]. Singlestranded oligonucleotide antagonists targeting ASBEL have been designed to improve the half-life of the lncRNA in the serum. lncRNA expression is also regulated by tyrosine kinase receptors (TKRs) and non-TKRs through the simultaneous action of multiple genes; more studies are required to identify unknown mechanisms for the simultaneous targeting of multiple genes [114]. Moreover, studies focusing on molecular mechanisms are needed to improve our understanding of how FDA-approved chemotherapeutic agents for malignant neoplasms exert their regulatory action through epigenetic mechanisms on TNBC. The expression level of lncRNAs is dysregulated by chromosomes 1 and 10 via an unknown mechanism, which also needs to be explored [112]. The co-localization of lncRNAs plays a major role in TNBC progression and endocrine-based resistance therapy; we need to consider co-expressed lncRNAs to identify possible strategies for better diagnosis and treatment options [115]. Some lncRNAs are used as biomarkers; high-throughput analysis of lncRNAs by next generation sequencing in TNBC cells should be conducted using cell lines and animal models to identify abnormally expressed lncRNAs in TNBC. lncRNA replacement therapy could potentially be used to restore tumour-suppressive lncRNAs [115]. A CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPRassociated protein 9)-based approach can be used to reprogram transcription regulatory network and immune regulations of lncRNAs [116][117][118] The co-expression patterns of lncRNAs with transcription and translation need to be further explored to identify genetic heterogeneity pathophysiology mechanisms for early diagnosis, drug discovery, and understanding the treatment response and drug resistance mechanisms in patients with TNBC.

Conclusions
Clinical management of patients with TNBC is difficult owing to aggressive tumour behaviour and histological heterogeneity. The biological behaviour of TNBC cells, including genetic and epigenetic regulation, is not fully understood. We need to discover novel molecular biomarkers and therapeutic targets for better treatment efficacy. Personalized therapy for TNBC patients is required to reduce TNBC progression. lncRNAs may prove to be very useful, as they play important roles in TNBC development and response to follow-up treatment. Considering the challenges of in vivo experimental designs, lncRNAs continue to be promising as biomarkers and potential therapeutic targets. Most lncRNAs exhibit low sequence conservation, which may limit the scope of efficacy. For instance, most lncRNAs are not common in humans or mice; therefore, loss-of-function experiments in mice are not feasible and positive clinical outcomes using this approach have remained limited. Accordingly, with the significant advances in the genetic study of lncRNAs, novel methods should be explored for diagnosis, therapy, and prognosis, but the potential clinical applications of lncRNAs are significant. RNAi is the most widely applied and efficient technology for targeting lncRNAs. To use lncRNAs in therapeutic settings, however, different technologies must be investigated, and more study is required. Compared to esiRNAs (endonuclease-made siRNA), conventional siRNAs show more off-target effects. Short hairpin RNA (shRNA), which is produced inside of cells, is another type of RNAi. When compared to esiRNAs, shRNAs have a significantly greater off-target effects and produce a silent response that may be temporary or sustained. Plasmid vectors containing shRNA or siRNA are used to transfect tumour cells. Therefore, toxicity and off-target effects are other limitations of the lncRNA delivery system. To overcome the limitations of current diagnosis and treatment strategies, additional research is required for the use of lncRNAs as diagnostic biomarkers and therapeutic targets in TNBC.

Conflicts of Interest:
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.